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Recombinant saccharomyces-fermentum-expressed hepatitis B surface antigen, production method of hepatitis B surface antigen, hepatitis B vaccine and production method of hepatitis B vaccine

A technology for recombining Saccharomyces cerevisiae and hepatitis B surface antigen, which is applied to the preparation methods of peptides, chemical instruments and methods, fermentation, etc., can solve the problems of high manual operation intensity, avoid manual operation, save equipment investment and maintenance costs, reduce The effect of labor intensity

Active Publication Date: 2013-10-02
SHENZHEN KANGTAI BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In this rough purification process, 8 to 10 large-capacity medium-speed centrifuges must be used, and the production operation time is 4 days. The manual operation is intensive, and the process cannot be completely completed in a closed system.

Method used

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  • Recombinant saccharomyces-fermentum-expressed hepatitis B surface antigen, production method of hepatitis B surface antigen, hepatitis B vaccine and production method of hepatitis B vaccine
  • Recombinant saccharomyces-fermentum-expressed hepatitis B surface antigen, production method of hepatitis B surface antigen, hepatitis B vaccine and production method of hepatitis B vaccine
  • Recombinant saccharomyces-fermentum-expressed hepatitis B surface antigen, production method of hepatitis B surface antigen, hepatitis B vaccine and production method of hepatitis B vaccine

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Embodiment 1

[0056] Take the working seed batch of recombinant Saccharomyces cerevisiae expressing HBsAg constructed by Merck Company in the United States with DNA recombination technology, and culture it in a 0.25L conical flask at 27-29°C for 17-19 hours, and in a 2L conical flask at 27-29°C. Cultivate for 17-19 hours, ferment and cultivate for 16-22 hours at 27-29°C in 70L seed tanks, ferment and cultivate for 44-48 hours at 27-29°C in 800L production tanks, and harvest the yeast.

[0057] After crushing the yeast by a high-pressure homogenizer, add an equal volume of phosphate buffer containing Triton X-100, and filter the yeast fragments with a filter membrane during the addition process to extract HBsAg.

[0058] Triton was removed by XAD-4 column to prepare Triton Antigen (PBP).

[0059] After loading macroporous silica gel with a pore diameter of 1000A and a particle size of 35 μm to 70 μm into a chromatography column to form a macroporous silica gel column, the macroporous silica ...

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Abstract

The invention discloses a production method of a recombinant saccharomyces-fermentum-expressed hepatitis B surface antigen. The method comprises the steps of: circularly removing triton from an extracted antigen by using XAD-4 column, so as to obtain triton-removed antigen sample liquid; loading macroporous silica gel with the pore size of 1,000 A and the particle size of 35-70 microns into a chromatographic column, and balancing by using phosphoric acid buffer solution with the pH of 7.6+ / -0.2; adjusting the pH of the triton-removed antigen sample liquid to be 7.6+ / -0.2 by using NaOH, then, loading a sample to the macroporous silica gel column; and cleaning impurities by using phosphoric acid buffer solution with the pH of 7.2+ / -0.2, carrying out eluting treatment on the impurity-cleaned macroporous silica gel column by using boric acid buffer solution with the pH of 8.7+ / -0.2 at the temperature of 47-49 DEG C, collecting eluate, and carrying out ultrafiltration and concentration on the eluate, thereby obtaining a clarified antigen. The invention further discloses the corresponding recombinant saccharomyces-fermentum-expressed hepatitis B surface antigen, a hepatitis B vaccine and a production method of the hepatitis B vaccine. The production methods have the advantages that the probability of product contamination is reduced greatly, the labor intensity for laborers is reduced, the equipment investment and repairing cost are reduced, the space occupied by equipment is reduced, and the production time is shortened.

Description

technical field [0001] The invention relates to biopharmaceutical technology, in particular to a hepatitis B surface antigen expressed by recombinant brewer's yeast and a production method thereof, a hepatitis B vaccine and a production method thereof. Background technique [0002] Hepatitis B virus (HBV) is a DNA virus. HBV infection is prevalent worldwide, but the prevalence of HBV infection varies greatly in different regions. According to the report of the World Health Organization, about 2 billion people in the world have been infected with HBV, of which 350 million people are chronic HBV infected people, and about 1 million people die every year from liver failure, liver cirrhosis and primary hepatocellular carcinoma caused by HBV infection . [0003] my country is a high-prevalence area of ​​hepatitis B, with an average of more than 500,000 cases reported each year. For many years, the incidence and incidence rate of hepatitis B have been at the forefront of the leg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K14/02C07K1/16A61K39/29A61P31/20A61P1/16
Inventor 甘建辉饶洪冲朱征宇梁全关敏肖坚
Owner SHENZHEN KANGTAI BIOLOGICAL PROD
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